Use of senicapoc for treatment of stroke

ABSTRACT

Neuroinflammation mediated by microglia and infiltrating peripheral immune cells is a major component of stroke pathophysiology. The calcium activated potassium channel K Ca 3.1 is expressed selectively in the injured CNS by microglia, and K Ca 3.1 function has been implicated in proinflammatory activation of microglia. K Ca 3.1 is further implicated in the pathophysiology of ischemia/reperfusion (stroke) related brain injury. Senicapoc, an investigational drug with a proven safety profile and shown to cross the blood-brain barrier, is a potent and selective K Ca 3.1 inhibitor that intervenes in the inflammation cascade that follows ischemia/reperfusion, and is a potential treatment for stroke.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is the U.S. National Stage of PCT International PatentApplication No. PCT/US18/16014 filed Jan. 30, 2018, which claims benefitof U.S. Provisional Patent Application No. 62/452,319 filed Jan. 30,2017 the disclosures of each of which are incorporated herein byreference in their entireties.

FIELD OF INVENTION

This invention pertains to the treatment of stroke with senicapoc.

BACKGROUND

Stroke is the leading cause of serious long-term disability and thefifth leading cause of death in the United States (Mozaffarian et al.,2015). Treatment options for stroke are few in number and limited inefficacy (Prabhakaran et al., 2015). The cellular response to acuteischemic stroke, particularly the response of immune cells, has beenstudied extensively and has been recently reviewed in detail (Iadecolaand Anrather, 2011; Macrez et al., 2011). The kinetics of post-strokeimmune reactions are critical in post-ischemic physiology and theconcept of a biphasic or multi-phasic response to brain ischemia is nowfavored (Hayakawa et al., 2010; Lo, 2008; Maki et al., 2013).Post-ischemic inflammation is characterized by a sequence of eventsinvolving the brain, its vessels, the circulating blood and lymphoidorgans. Many of these processes play a central role inpreconditioning-mediated neuroprotection (McDonough and Weinstein,2016).

Microglia are central nervous system (CNS)-resident immune cells(Benarroch, 2013; Michell-Robinson et al., 2015) derived from yolk sacmacrophages that enter the CNS during early development and maintainthemselves as a distinct population from circulating monocytes (Ginhouxet al., 2010). Microglia contribute to the maintenance of brainhomeostasis by pruning synapses, clearing dead or dying cells as well asproviding trophic support to other cells (Zuchero and Barres, 2015).These functions suggest that microglia play a critical role in thenormal physiology and development of the CNS (Jha et al., 2015).Microglia play a significant role in the neuroinflammatory response toischemia (Weinstein et al., 2010). The expression of Toll-like receptors(TLRs) and other pattern recognition receptors by microglia enables themto identify pathogens and upregulate a unique profile of effector immunecytokines and chemokines in response to a wide range of stimuli (vanRossum and Hanisch, 2004). Most abundantly expressed by microglia isTLR4, and both endogenous and exogenous TLR4 agonists potently activateclassical pro-inflammatory responses in microglia (Dave et al., 2006;van Rossum and Hanisch, 2004).

Although microglial activation has typically been considered apro-inflammatory process, recent publications suggest that microgliacould play a protective role in stroke (Lalancette-Hebert et al., 2007;Nedergaard and Dirnagl, 2005) through multiple mechanisms such asmetabolic and physiological support of neurons (van Rossum and Hanisch,2004), production of trophic factors (Nedergaard and Dirnagl, 2005),autophagy of damaged and repair of lesioned tissue (Cunningham et al.,2005). Microglia are the first responders to ischemic injury, activatingbefore peripheral monocytes/macrophages infiltrate the CNS (Umekawa etal., 2015). Ischemia induces robust increases in microglia cell number(Denes et al., 2007; McDonough and Weinstein, 2016) and proliferation(Denes et al., 2007). Pharmacologic or genetic ablation of microgliainfluences outcome in multiple rodent models of stroke(Lalancette-Hebert et al., 2007; Szalay et al., 2016). These findingshave provided strong evidence to support a key role for innate immunesignaling and microglia in both ischemia-induced injury andneuroprotection.

The effects of microglial activation and its role in post-ischemicinflammation is shown schematically in FIG. 1 (Staal et al., NeurochemRes. 2017). As shown in FIG. 1, astrocytes (AS) provide trophic supportto neurons (N) through multiple mechanisms and secrete transforminggrowth factor β (TGFβ), which is reparative to endothelial cells (EC).Both microglia (MG) and astrocytes secrete pro-inflammatory cytokines(tumor necrosis factor α (TNFα), IL-1β, IL-6, IL-17) in response toischemia. Neurons also signal via fractalkine (CX3CL1) to microgliawhich express cognate receptor CX3CR1. Both astrocytes and peripheralimmune cells (PIC) are potential sources of type 1 inteferons (IFNα, β)that signal to microglia via IFNAR, triggering transcription ofinterferon stimulated genes (ISGs). ISG protein products may enhanceoligodendrocyte (OL) viability in the setting of prolonged ischemia andin turn increase axonal integrity in white matter. The latter may limitlong-term ischemia-induced injury to neural networks and protect thewhite matter-based connectome. ECs and other cells release dangerassociated molecular patterns (DAMPs), such as fibronectin, highmobility group box 1 (HMGB1), peroxiredoxin (PRX) and heat shockproteins (HSPs) that are endogenous ligands for numerous TLRs. PICs arecapable of secreting many different cytokines, which have effects onmultiple cell types.

Thus, pharmaco-therapeutics that can specifically modulate microglialgene expression and phenotype in the context of ischemia may be able toeffectively skew the neuroimmune response in a direction that is morefavorable to both neuronal survival and axonal/white matter integrity.

The calcium activated potassium channel K_(Ca)3.1 is constitutivelyexpressed in the CNS by cerebrovascular cells. It is also expressed bymicroglia following injury to the CNS (Chen et al., 2015). K_(Ca)3.1leads to potassium efflux thereby increasing the driving force for Ca²⁺entry, and subsequently affecting Ca²⁺ dependent immune mechanisms. Ithas been shown that microglia in vitro express K_(Ca)3.1 and that itsinhibition reduces production and release of nitric oxide (NO) andinterleukin 1β (IL-1β) from appropriately stimulated microglia (Dale etal., 2016) (FIG. 1). Other studies have shown that inhibition ofK_(Ca)3.1 reduces microglial synthesis of enzymes involved in productionof eicosanoids (COX-2) and nitric oxide (iNOS) (Nguyen et al., 2016).Accordingly, inhibition of K_(Ca)3.1 is expected to have a broad rangeof anti-inflammatory effects.

Several inhibitors of K_(Ca)3.1 have been reported (Wulff and Castle,2010; Wulff et al., 2007). However, early inhibitors lacked potency andselectivity and were hampered by safety concerns (Suzuki et al., 2000;Wulff and Castle, 2010; Zhang et al., 2002).

TRAM-34 was described as a selective inhibitor of K_(Ca)3.1 with goodpotency and good CNS penetration. TRAM-34 potently inhibits K_(Ca)3.1channels with an IC₅₀ of 20 nM in recombinant cell lines and has noeffect on cytochrome P450-dependent enzymes (Wulff et al., 2000). It hasbeen used to investigate the physiology of K_(Ca)3.1 channels in immunecells and the involvement of K_(Ca)3.1 channels in several CNSdisorders, including multiple sclerosis (Reich et al., 2005), opticnerve transection (Kaushal et al., 2007), spinal cord injury (Bouhy etal., 2011), ischemic stroke (Chen et al., 2011; Chen et al., 2015), andglioblastoma multiforme (D'Alessandro et al., 2013). Wulff andcolleagues evaluated TRAM-34 in a rat model of ischemic stroke (Chen etal., 2011). After administration of TRAM-34 at 40 mg/kg intraperitoneal(i.p.), plasma and brain concentrations reached ˜1 μmol/L at 8 hours,dropping to 0.4 μmol/L by 12 hours. Free plasma concentrations weredetermined to be approximately 2%. From these data, it is estimated thatthe plasma and brain concentrations are 20 nM and 8 nM, respectively, at12 hours (before the second dose). Thus, when given i.p. the TRAM-34concentrations are at or near the IC₅₀ values for K_(Ca)3.1 inhibition.The high doses needed to achieve concentrations above IC₅₀ values,however, suggested that bioavailability for TRAM-34 is a significantissue.

Unbound CNS levels of the TRAM-34 are not much higher than the IC₅₀ forK_(Ca)3.1 inhibition in microglia in vitro and the t_(1/2) suggests thatCNS K_(Ca)3.1 inhibition is only achieved for a few hours afteradministration. While it is always challenging to develop a CNSpenetrant drug able to provide 24-hour coverage even with a multipledosing paradigm, there is significant room for improvement in druglevels achieved as well as t_(1/2). Modulating the neuroimmune response,and the microglial/macrophage phenotype in particular, is an attractivetarget in acute ischemic stroke therapy in part because this responseevolves gradually over days to weeks, whereas many previously targetedphysiological phenomena in stroke, such as glutamate-dependentexcitotoxicity for example, tend to occur rapidly (minutes to hoursafter stroke onset) (Dirnagl, 2012). Thus, targeting the neuroimmuneresponse in stroke offers a broader temporal therapeutic window andcould translate to therapies beyond the current three to six-hour timewindow. Administration of TRAM-34 at both 10 and 40 mg/kg i.p.significantly attenuated post stroke infarct volume and neuronal loss.TRAM-34 also improved the neurological deficit score and significantlyreduced the extent of microglial ED1 staining. Especially promising wasthe finding that TRAM-34 improved the outcome in this model of strokeeven when given 12 hours after the ischemic insult. Currentpharmacologic treatments for acute ischemic stroke need to be givenwithin 3-4.5 hours (Prabhakaran et al., 2015), a temporal challenge thatseverely limits the reach of currently available therapies.

While TRAM-34 showed selectivity for K_(Ca)3.1 over othercalcium-activated potassium channels (Wulff et al., 2000), it might haveinhibited additional targets, confounding the interpretation of anyresults (Schilling and Eder, 2007). Schilling and Eder have demonstratedthat TRAM-34 blocks non-selective cation current in primary microgliastimulated with lysophosphatidylcholine (LPC) with an IC₅₀ that wassimilar to its IC₅₀ for K_(Ca)3.1 channels (Schilling and Eder, 2007).Furthermore, another presumed K_(Ca)3.1 blocker, charybdotoxin, had noeffect on LPC signals (Schilling and Eder, 2007). Hence, TRAM-34 maymodulate immune cell function by a mechanism unrelated to K_(Ca)3.1inhibition. Furthermore, it has recently been demonstrated that TRAM-34still inhibits some cytochrome P450 isoforms, namely human CYP2B6,CYP2C19 and CYP3A4 with IC₅₀ values in the low micromolar range (Agarwalet al., 2013).

In addition, TRAM-34 shows metabolic instability and has a shorthalf-life (˜2 hours in rats and primates) potentially complicatingchronic dosing (Maezawa et al., 2012). Thus, although TRAM-34 is avaluable experimental and potentially effective therapeutic agent, ithas issues that may confound interpretation of mechanism in pre-clinicalmodels and may limit its clinical utility.

Another reported K_(Ca)3.1 inhibitor is NS6180,(4-[[3-(trifluoromethyl)phenyl]methyl]-2H-1,4-benzothiazin-3(4H)-one)(Strøbæk, et al., 2013). NS6180 was reported to have similar potency andselectivity to TRAM-34.

An alternative K_(Ca)3.1 inhibitor is senicapoc (ICA-17043), a potent,CNS penetrant inhibitor with improved stability and selectivity vsTRAM-34 (Dale et al., 2016; Strøbæk, et al., 2013; Schilling and Eder,2007). Senicapoc is an experimental drug described in U.S. Pat. No.6,288,122. Senicapoc has previously been investigated for the treatmentof sickle cell anemia (Ataga et al. 2008) and malaria (Tubman et al.2016). However, a phase III clinical trial for sickle cell disease foundthat despite improvements in hematological parameters, there was noimprovement in sickle cell painful crises observed, so developmentactivities were halted (Ataga et al., 2011). Significantly, the sicklecell clinical trial was not terminated for safety or toxicity reasons.

SUMMARY OF THE INVENTION

In view of the demonstrated selective inhibition of on K_(Ca)3.1 bysenicapoc, concurrent reduction of nitric oxide and regulation on Ca⁺⁺signaling, and the ability of senicapoc to cross the blood-brainbarrier, senicapoc exerts a neuroprotective effect and can prevent orreduce stroke or ischemia-induced injury in a patient at risk for strokeor ischemia-induced injury, or in acute ischemic stroke patients.Moreover, because of the anti-inflammatory effect of inhibition ofK_(Ca)3.1, senicapoc is useful for treating a patient suffering fromstroke or ischemic injury and reducing the neuroinflammation associatedwith these conditions.

Accordingly, in an embodiment, a method is provided of preventing ortreating stroke by the administration of senicapoc to a patient atimmediate risk for stroke or ischemia-induced injury or who is activelysuffering from acute stroke or ischemia-induced injury. In anembodiment, senicapoc is used in the manufacture of a medicament for theprevention or treatment of stroke by the administration of senicapoc toa patient at risk for stroke or ischemia-induced injury or sufferingfrom stroke or ischemia-induced injury. In an embodiment, an oral dosageform of senicapoc is provided for use in preventing or treating strokeby the administration of senicapoc to a patient at risk for stroke orischemia-induced injury or suffering from stroke or ischemia-inducedinjury.

DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic showing key neuroimmune pathways and interactionsbetween cells of the CNS in ischemic inflammation and the points ofsenicapoc activity.

FIG. 2. Effect of K_(Ca)3.1 inhibition on NO and regulation of Ca⁺⁺signaling. All downstream effects in microglia (shown in green) andcerebrovascular endothelial cells (shown in red) are attenuated bysenicapoc electrophysiology.

FIG. 3. K_(Ca)3.1 electrophysiology in rat microglial cells exposed tocontrol or senicapoc. K⁺ currents were recorded from primary microgliaby using either (A) depolarizing steps or (B) a voltage ramp protocol.In both paradigms, the currents reversed at 0 mV which was theequilibrium potential for K⁺. Senicapoc dose dependently inhibited asignificant part of the K⁺ current.

FIG. 4. Effect of K_(Ca)3.1 inhibition on NO and IL-1β release fromprimary microglia. (A) Primary rat cortical microglia were pre-treatedwith vehicle or senicapoc at concentrations indicated for 30 minutesfollowed by addition of lipopolysaccharide (LPS) (3 EU/ml) and incubatedfor a total of 24 hours. Senicapoc inhibited the production of NO (asmeasured by its metabolite, nitrite) with an EC₅₀ of 0.9 nM as shown ina representative experiment (n=3). (B) Primary rat cortical microgliawere incubated with LPS (3 EU/ml) for 24 hours to induce expression ofIL-1. Senicapoc dose dependently inhibited IL-1 release from primarymicroglia with an IC₅₀ of 1.3 nM (n=3).

DETAILED DESCRIPTION

Stroke is the leading cause of serious long-term disability and thefifth leading cause of death in the United States. Treatment options forstroke are limited and have limited efficacy. Neuroinflammation mediatedby microglia and infiltrating peripheral immune cells is a majorcomponent of stroke pathophysiology. Interfering with the inflammationcascade after stroke holds the promise to modulate stroke outcome. Thecalcium activated potassium channel K_(Ca)3.1 is expressed selectivelyin the injured CNS by microglia. K_(Ca)3.1 function has been implicatedin pro-inflammatory activation of microglia and there is recentliterature suggesting that this channel is important in thepathophysiology of ischemia/reperfusion (stroke) related brain injury.Accordingly, senicapoc, a K_(Ca)3.1 inhibitor, may intervene in theinflammation cascade that follows ischemia/reperfusion and limit thedamage caused by stroke or other ischemic injury.

Senicapoc attenuates pro-inflammatory responses in microglia (reducingrelease of cytokines and nitric oxide) and in epithelial cellsattenuating ischemia-induced disruption of the blood-brain barrier (BBB)(FIG. 1) (Staal et al., Neurochem Res. 2017). By modulating elements ofthe microglial and epithelial cells response to ischemia, senicapoc mayinfluence the neural environment indirectly in a number of ways, forexample by enhancing white matter integrity as shown in FIG. 1.

The ability of senicapoc to inhibit the potassium channel K_(Ca)3.1 isdiscussed in detail in our co-pending patent application PCT/US17/57930,filed Oct. 23, 2017.

K_(Ca)3.1 is highly expressed on microglia in vitro (Kaushal et al.,2007). The effect of senicapoc was evaluated on microglial K⁺ currentselicited by either depolarizing steps (FIG. 3A) or a voltage rampprotocol (FIG. 3B) using automated patch clamp analysis. Senicapoc dosedependently (10, 100, 300 and 1000 nM) inhibited the microglial K⁺current although not completely (FIG. 3A) with an IC₅₀ of 10 nM. Thisvalue is in close agreement with the IC₅₀ value (10 nM) generated bypatch-clamp studies on CHO-K_(Ca)3.1 cells. Some residual K⁺ currentstill remained which was most likely not K_(Ca)3.1-sensitive (Kettenmannet al., 2011).

To show the inhibition of nitric oxide and IL-1β, primary rat corticalmicroglia were incubated with either vehicle or senicapoc for 30 minutesprior to the addition of vehicle or ultrapure LPS (3 EU/ml) to stimulateiNOS expression and NO release. After 24 hours, media was assayed fornitrite (stable metabolite of NO). Senicapoc dose dependently inhibitedthe release of NO from LPS-treated microglia with an average IC₅₀ of 39nM (FIG. 4A), in agreement with previous studies (Kaushal et al., 2007;Khanna et al., 2001). Primary rat cortical microglia were also treatedwith LPS (3 EU/ml, 3 hours) to stimulate the production of pro-IL-1β.Next, vehicle or senicapoc were added and incubated for an additional 30minutes followed by the addition of BzATP (1 mM) to activate P2X7receptors and trigger the activation of caspase 1, its cleavagepro-IL-1l and the release of the liberated IL-1 (another 30 minutes).Senicapoc dose dependently inhibited IL-1 release from primary microgliawith an IC₅₀ of 15 nM (FIG. 4B).

Unlike TRAM-34, senicapoc has no known off target effects atconcentrations that block K_(Ca)3.1 (Staal et al., 2017). It also doesnot suffer from metabolic instability or effects on cytochrome P450.Most importantly, senicapoc has been tested in humans in clinical trialswithout any significant side effects. The finding that senicapoc is alsoCNS penetrant opens up its use for CNS indications.

While many devastating neurological and perhaps psychiatric diseasescould be potentially treated by senicapoc, the studies of selectiveinhibitor of K_(Ca)3.1 with high potency and good CNS penetration as atreatment for stroke lay a foundation for the use of senicapoc on strokepatients.

Finding a treatment for stroke that can be given beyond the narrowtherapeutic window of current treatments would be a major advance. Thedata on efficacy of the K_(Ca)3.1 inhibitor, TRAM-34, outside of thisnarrow therapeutic window suggests that inhibition of K_(Ca)3.1 couldbecome a promising treatment strategy in acute stroke. The potent andselective K_(Ca)3.1 inhibitor senicapoc (IC₅₀ of 11 nM) was initiallydeveloped for the treatment of sickle cell anemia (Ataga et al., 2006;Ataga et al., 2011; Ataga et al., 2008; Ataga and Stocker, 2009). Thedrug was well tolerated in Phase 1 clinical trials in both healthyvolunteers and in patients with sickle cell disease (Ataga et al., 2006;Ataga et al., 2011). In a double-blind placebo controlled Phase 2 study,senicapoc (at 10 mg/day) reduced hemolysis and significantly increasedhematocrit and hemoglobin levels in patients with sickle cell disease(Ataga et al., 2008). In a subsequent Phase 3 trial, senicapoc wastested for its effects on vaso-occlusive pain crisis (Ataga et al.,2011). However, despite properly engaging erythrocyte K_(Ca)3.1,reducing hemolysis and increasing hemoglobin and hematocrit levels,senicapoc had no effect on pain outcome measures and the trial wasterminated (Ataga et al., 2011). While this was disappointing, it isimportant to point out that the drug did what it was supposed to do on amolecular and cellular level. The clinical trial failed because theoutcome measure chosen were distal to the proposed mode of action andperhaps not completely dependent on this mechanism.

Importantly, senicapoc has been shown to cross the blood-brain barrier.While the peripheral pharmacokinetics of senicapoc have been describedin detail (McNaughton-Smith et al., 2008), the ability of senicapoc tocross the blood-brain barrier has only recently been reported (Staal etal., 2017). After 10 mg/kg oral dosing in rats, senicapoc achieved freeplasma concentrations of 17 and 65 nM and free brain concentrations of37 and 136 nM at one and four hours post-dose, respectively.Cerebrospinal fluid (CSF) concentrations were determined to be 25 and121 nM at one and four hours post-dosing which are in-line with the freebrain concentrations. These data suggest that senicapoc achieves CNSconcentrations greater than its IC₅₀ value for K_(Ca)3.1 channels (11nM) and thus should be sufficient to inhibit it (McNaughton-Smith etal., 2008). Furthermore, senicapoc achieves free brain concentrationsseveral fold higher than TRAM-34.

In the same study, senicapoc's selectivity was assessed in a screen of˜70 additional neuronal drug targets (50 neuronal receptors, 8 enzymes,5 transporters and 7 ion channels) (Staal et al., 2017). None of thetargets tested was inhibited by senicapoc at 1 μM, providing additionalevidence that senicapoc is selective for K_(Ca)3.1 channels. In vivo,senicapoc was tested in the chronic constriction injury model ofneuropathic pain (Bennett and Xie, 1988). Senicapoc dose dependently(10, 30 and 100 mg/kg p.o.) attenuated the mechanical hypersensitivityinduced by the peripheral nerve injury, although only the highest dosewas significant (Staal et al., 2017). Furthermore, in contrast toreported locomotor effects in kcnn4^(−/−) mice (Lambertsen et al., 2012)that have no functional K_(Ca)3.1, the authors did not observe anysignificant impact of senicapoc on locomotor activity (Staal et al.,2017). While the study does not shed light on the cell types in the CNSthat express K_(Ca)3.1, it clearly demonstrates that senicapoc wasefficacious in ameliorating pain behaviors in rats with peripheral nerveinjury and these conclusions were supported by the free drugconcentrations attained in plasma, brain and CSF.

Numerous active cellular processes and complex cellular interactionscontribute to the resolution of post-ischemic inflammation. senicapocameliorated pain behaviors in a model of neuropathic pain (Staal et al.,2017). Since experimental surgery-related inflammation is resolved sevendays after the animals are tested, it supported the hypothesis that theefficacy was mediated by inhibition of K_(Ca)3.1 on microglia in thespinal cord or brain rather than peripheral immune cells. In addition tothe prior studies in rats, we report here the ability of senicapoc topenetrate the CNS in mice (see Table 1). The data were similar to thosein rats with senicapoc reaching higher levels in brain than plasma andshowing a similar t_(1/2) demonstrating that senicapoc readily crossedthe blood brain barrier and achieved concentrations well above the IC₅₀.Based on the rat and mouse pharmacokinetic data CNS penetrance in humansseems promising.

To address in vivo side effects of senicapoc, the most relevant beingsedation in pain models, the effect of the drug on rat locomotoractivity was tested (Staal et al., 2017). The results showed thatsenicapoc did not alter activity at doses required for efficacy in thechronic constriction injury model of neuropathic pain. The data suggeststhat K_(Ca)3.1 inhibition has few adverse effects. The significance ofthese pre-clinical findings is enhanced by the human clinical trialsthat demonstrated that senicapoc is safe and has a low incidence of sideeffects.

Based on the animal studies, the major drawback to both TRAM-34 andsenicapoc is the short half-life (see Table 1). In contrast to thepreclinical studies in rodents, clinical trials showed an unexpected t %of 23 days in humans. This raises the question whether senicapoccovalently binds to plasma proteins whose t % is approximately 21 dayswhich is significantly longer than that of the unbound drug. It isimportant to note that the potential covalent protein binding, shouldnot impact the ability of senicapoc to penetrate the CNS, although itwould make dose titration more complex.

To date, the only CNS disease model in which senicapoc has beenevaluated is the chronic constriction injury model of neuropathic pain(Staal et al., 2017).

TABLE 1 Pharmacokinetics of TRAM-34, NS6180 and senicapoc *IC₅₀ reportedare for human KCa3.1 expressed in recombinant cells. All data are forcompounds dosed p.o. TRAM- 34 NS1680 Senicapoc Senicapoc MolecularWeight 345 323 323 (g/mol) Species Rat Mouse Dose (mg/kg) 10 10 10 10 T½(hours) ~2 3.8 1 3.1 T_(max) (hours) 0.5-1 4 0.33 In-vitro IC₅₀ (nM)* 209.4 hu 12 hu (Wulff et al 2000) C_(max)-Total Plasma ~2500 186 2,400(Staal 709 (nM) et al., 2017) Brain ~2500 17,000 (Staal et al., 2017) %unbound Plasma 2 2.7 (Staal et al., 1.5 2017) C_(max)- Brain 0.8 (Staalet al, 0.8 Unbound 2017) (nM) Plasma 50 65 C_(max)- Brain 50 136 Unbound(nM) CSF 121 Reference Chen et Strøbæk McNaughton- Previously al., etal, 2013 Smith, 2008 unpublished 2011 data JCBM hu: tested in vitro inthe human receptor.Compounds and Formulations

In an embodiment, senicapoc may be formulated as an oral pharmaceuticalformulation product for use in the prevention or treatment of stroke orischemic injury as provided herein.

Oral dosage forms include tablets, capsules, and powders for dissolutionor suspension in a drink. Such tablets and capsules may be formulated byany of various methods known in the art and may include at least oneexcipient.

Senicapoc has previously been developed exclusively as an oralformulation, but for stroke patients, an intravenous formulation may bedesirable.

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The invention claimed is:
 1. A method of inhibition of calcium activatedpotassium channel K_(ca)3.1 on microglia in the brain, by theadministration of an amount of senicapoc sufficient to achieve acerebrospinal fluid (CSF) concentration of 11 nM or greater.
 2. A methodof inhibition of calcium activated potassium channel K_(ca)3.1 onmicroglia in the brain in a patient experiencing ischemia/reperfusionbrain injury by the administration of an amount of senicapoc sufficientto achieve a cerebrospinal fluid (CSF) concentration of 11 nM orgreater.
 3. A method of treating ischemia/reperfusion brain injury,comprising the inhibition of calcium activated potassium channelK_(ca)3.1 on microglia in the brain, by the administration to a patientexperiencing ischemia/reperfusion brain injury an amount of senicapocsufficient to achieve a cerebrospinal fluid (CSF) concentration of 11 nMor greater.